The 3DNA® DNA dendrimer is a proprietary dendritic molecule comprised solely of DNA. As a class, dendrimers are complex, highly branched molecules built from interconnected natural or synthetic monomeric subunits. A DNA dendrimer is constructed from DNA monomers, each of which is made from two DNA strands that share a region of sequence complementarity located in the central portion of each strand (FIG. 1). Monomers are combined during the manufacturing process to prepare DNA dendrimers of different sizes and shapes (FIG. 2). In order to prevent DNA dendrimers from “falling apart” over time, chemical “spot welds” are sometimes added to the growing assembly during the process using UV light via the intercalation and activation of psoralen cross-linkers. Dendrimers have been historically purified according to their size and molecular weight on denaturing sucrose gradients after ultracentrifugation.
DNA dendrimers have previously been used in membrane based assays, specifically for the detection of nucleic acids and proteins non-covalently immobilized to various membrane substrates, including nitrocellulose and nylon. These assays typically required the use of dendrimers specifically derivatized to contain targeting or binding moieties specific for the target analyte, and typically required several hours to overnight for optimal binding. Improvement of sensitivity from signal amplification ranged from 5 to 500 fold over the non-dendrimer version of the assay.
DNA dendrimers have also been shown to be useful as signal amplifiers in a number of other applications, including nucleic acid (DNA/RNA) microarrays, ELISAs, ELOSAs, bead based immunoassays, protein arrays and other similar assays. These assays are all characterized by the immobilization of the analyte or target material to a substrate either directly via a non-covalent or a covalent binding process, or indirectly via the binding to a previously immobilized ligand, which generally required several steps prior to or during the assay process. DNA dendrimers containing up to hundreds of label moieties would then bind either directly or indirectly to the analyte via a targeting device which would simultaneously directly bind to both the analyte (or indirectly to a ligand bound to the analyte) and the dendrimer, thereby creating a bridge between the multi-labeled dendrimer and the analyte. Signal was generated in these assays either directly from label moieties on the dendrimer (e.g. fluorescent dyes) or indirectly via the binding of signaling moieties to binding sites on the dendrimer (e.g. streptavidin-HRP binding to dendrimer bound biotins).
Therefore, there is a need for devices and methods that overcome the limitations of the current technologies and methodologies, especially for more sensitive lateral flow assays. The present invention addresses these and other related needs.